Extended Data Fig. 1: Tyrosine sulfation is a novel type of histone modification.
From: Histone tyrosine sulfation by SULT1B1 regulates H4R3me2a and gene transcription
(a), Identification of H3Y99phos peptide in HepG2 cells. MS/MS spectra of a phosphorylated histone H3 peptide (H3Y99phos) was derived from HepG2 cells (in vivo). The b ion refers to the N-terminal parts of the peptide, and the y ion refers to the C-terminal parts of the peptide. Data represent two independent experiments. (b) and (c), validation of H3Y99phos peptide in HepG2 cells. (b), The chromatographic elution profile from HPLC–MS/MS analysis of H3K99phos peptides derived from cultured HepG2 cells (in vivo) and the synthetic counterparts; (c), MS/MS spectra of a phosphorylated histone peptide (H3Y99phos) derived from HepG2 cells and its synthetic counterpart. The b ion refers to the N-terminal parts of the peptide, and the y ion refers to the C-terminal parts of the peptide. Data represent two independent experiments. (d) to (g), Generation of specific antibody against human H3Y99sulf. (d) and (e), H3Y99sulf-antibody specifically recognizes H3Y99sulf peptide. The binding of H3Y99sulf-antibody to H3Y99 control peptide (CEASEAYLVGLF), H3Y99phos-peptide (CEASEA-Y (phos)-LVGLFR), and H3Y99sulf-peptide (CEASEA-Y (Sulf)-LVGLFR) were tested by performing dot-blotting assay. Only H3Y99sulf peptide can block the application of H3Y99sulf-antibody in the dot-blotting assay; (f) and (g), H3Y99sulf peptide blocks the application of H3Y99sulf-antibody in western blotting and immunoprecipitation assays. H3Y99sulf-antibody was incubated with or without control peptide (10 μg/ml), H3Y99phos-peptide (10 μg/ml) and H3Y99sulf-peptide (10 μg/ml) for 24 hours before the western blotting and immunoprecipitation assays. Data represent three independent experiments. (h), Testing non-specific binding of H3Y99sulf-antibody. A microarray that contains histone peptides was used to test whether the developed H3Y99sulf-antibody read histone regions without H3Y99sulf. The antibody concentration is identical as used in the immunoblotting assays. Data represent one independent experiment.
